Production of Rabbit Monoclonal Antibodies Againts Mouse Embryonic Stem Cells and Identification of

Pluripotency-associated Surface Antigen

Dewi Satwika

116090100011006

PROGRAM PASCA SARJANA JURUSAN BIOLOGI

FAKULTAS MATEMATIKA DAN PENGETAHUAN ALAM

UNIVERSITAS BRAWIJAYA

detection of antigen

Embryonic stem cells (ES cells)

potential for regenerative medicinepluripotent stem cells

ES cells surface moleculesgenomics

proteomics

Monoclonal antibody Efficient method for surface marker identification

Kohler and milsteinMus muscullus

hibridoma

Smaller Ab repertoire

Immunotolerant to cell surface Ag on mouse ES cells

Potential to recogniize new

surface molecule on

mouse ES cellsRabbit New Zealend

larger antibody repertoire

higher affinity andspecificity in recognizing

conformational and modified epitopes

?

METHODSInjected

subcutaneously with 108 mES D3 cell line

The mES cell lines D3 were cultured on mitomycin C-treated mouse embryonic fibroblasts (MEFs)3 months old

Resuspended in 1 ml PBS

emulsified with CFA in a 1:1 (v/v) ratio

Booster using mES cell suspension emulsified in IFA

injected intravenously with 108 mES cells suspended in 1 ml of

PBS

serum collection

1 week

Sacrifice

Spleen240E-1 cell

line

Added PEG to fuse

hibridoma

mES cells culture in MEF

Supernatant hibridoma

centrifuge

FITC conjugatedgoat anti-rabbit

sec. Ab

clone

2 weeks

clone Positive clone

propagated

Culture supernatant Cells lysate

5×108 F9 EC cells by ultrasonication in

lysis buffer

Culture supernatant

Protein-A conjugated resin

Putative antigens eluted with a 2.5 mM citrate solution

cSDS PAGE

Fluoresence microscopy cells

Fixed in 4% PFA

Blocked with blocking solution

Staining primary and second Ab

Western blotting

Flowcytometry

RESULT

Fig. 1. Staining patterns of different rMabs on mES D3 cells and MEFs.

After subcloning, 240 remained positive. Six monoclones of each subclone plate were expanded and cryopreserved.

These antibodies can be divided into four major types based on the cellular locations of their targeted antigens: membrane (A), extracellular matrix (B), nucleus (C) and cytoplasm (or cytoskeleton) (D).

Fig. 2. ICC staining using the 20 rMabs.

7 antibodies showed nuclear or diffused staining while 13 were cytosolic/membraneous.

Twenty Mab that were able to bind to mES cells. These Ab

showed different staining patterns.

All but two antibodies stained positively on

mES cells

Positively stained by three antibodies, ZjuESrMab3,

ZjuESrMab29 and ZjuESrMab61, decreased more than 50% upon mES

cell differentiation

10 antibodies targeted the extracellular epitopes of cell surface proteins for more than 5% of the mES cells

Fig. 3. Flow cytometry analysis using the 20 rabbit monoclonal antibodies. A: Staining of monoclonal antibodies on mES D3 cells. B: Staining of monoclonal antibodies on mES D3 cells that were spontaneously differentiated for 3 days

From 20 positively rMabs

ZjuESrMab29 and ZjuESrMab61 did not detect any protein in the western blot analysis, suggesting that they might target conformational epitopes on live mES cells.

one specifically stained band of approximately 42 KDa was observed for ZjuESrMab29

LC-MS/MS analysis identified the protein as GM-CSFR α.

ICC staining with ZjuESrMab29 faded after three days of spontaneous differentiation of mES cells cultured in a monolayer.The percentage of positive cells decreased to about 1%.

A small increase in staining was detected after nine days of monolayer differentiation The number of positively stained cells decreased to about 2% after four days of differentiation into embryoid bodies (Ebs)

Immunohistochemical analysis showed that ZjuESrMab29 stained strongly in the testis, stained weakly in the restricted, perivascular regions of the kidney and spleen and did not stain in the liver,

heart, brain or lung

Immunohistochemical staining of adult mouse tissues with ZjuESrMab29. ZjuESrMab29 stained strongly in the testis, stained weakly in the restricted regions of the kidney and spleen and did not stain in the liver, heart, brain or lung.

The expression of GM-CSFR α in the testis may serve as a marker of ES

cells and GS cells.

expression of GM-CSFR α decreases upon mES cell differentiation and is restricted to adult tissues,

suggesting that GM-CSFR α could serve as a new pluripotency- associated surface marker for mES

cells.

Thank You